Isolation and Purification of Vertebrate DNAs

Abstract

Isolation of DNA from surrounding tissues is a crucial initial step in any molecular biological investigation of organisms. DNA in cells is complexed with numerous proteins and exists in an environment saturated with a whole host of other biomolecules. This chapter presents two alternative methods for the isolation of total cellular DNA from vertebrate tissues. Method 1 is a variation on the standard phenol extraction procedure (1), which is still the most widely used technique for obtaining DNA from animal tissue. Method 2 relies on the use of a saturated salt solution and avoids the need to handle hazardous phenol preparations. Both are suitable for a whole range of vertebrate tissues and provide DNA suitable for most commonly used further manipulations, including cloning and library construction, polymerase chain reaction (PCR) (of both nuclear and mitochondrial regions), multilocus finger-printing, and analysis of particular genes and gene families (e.g., ribosomal DNA) through restriction enzyme digestion and Southern blotting. Also included is a protocol that relies on boiling in the presence of Chelex resin to provide DNA suitable for use directly in PCR analysis (2). This procedure has proved useful when dealing with very small quantities of starting material, in forensics, for example, and is relatively quick and easy to perform. The method given for the isolation of purified mitochondrial DNA relies on differential centrifugation of intact mitochondria followed by phenol extraction and is a modification of that given by Powell and Zuniga (3) and Jones et al. (4). The technique avoids the use of lengthy ultracentrifugation procedures (5) and expensive cesium chloride gradients, and can provide mitochondrial DNA of sufficient quantity and purity for use in restriction fragment length polymorphism studies using the sensitive silver-staining visualization technique (6,7). Tissue choice and storage are crucial considerations in any study, and these are considered in the Notes section.